Caloric restriction (CR) extends life-span and delays diseases in animal models. This remarkable observation has been difficult to translate into human health benefits, for two primary reasons. First, a life-time of food deprivation and reduced body weight is not practical for most people;and, second, it has not been easy to identify the underlying metabolic and molecular signals responsible for health benefits in rodent models when each experiment takes up to 3 years (to show that animals live longer). We recently applied a biomarker- based strategy, using highly sensitive, stable isotope-mass spectrometric measurements of fluxes through metabolic pathways in vivo as outcome measures, and looked for dietary regimens that may mimic the benefits of CR without requiring net negative energy balance, to address these limitations. Our studies have shown that intermittent feeding (IF) regimens such as modifed alternate-day fasting (mADF) reproduce many of the benefits of true CR in mice, without weight loss or change in body composition. Biomarkers that respond to CR and certain mADF regimens include global cell proliferation (mammary, prostate, skin, liver, lymphocytes), adipose tissue dynamics and fat distribution, insulin sensitivity, and vascular smooth muscle cell proliferation. We also observed similarities between IF and CR regimens with regard to striking cyclicity of whole-body fuel utilization and food intake, indicating that CR is itself a form of IF. In this project, we will use rapidly responsive biomarkers of disease risk to explore potentially more feasible dietary regimens and to identify underlying metabolic and molecular signals responsible for benefits. Specific aims are, 1) establish the pattern of IF required to reproduce the effects of CR (duration of fasting, feeding frequency, macronutrient content). 2) Identify metabolic signals (e.g., NADH/NAD, glycogen, fatty acid oxidation products) associated with effective IF regimens. 3) Identify changes in molecular signaling pathways (e.g., sirtuin pathway gene expression and target acetylation, IGF-1/insulin axis, PGC-1, nuclear factors) potentially mediating changes in biomarkers, including studies in gene knock-out mice (liver-specific SIRT3, IGF-1 receptor). 4) Evaluate combinations of exercise or CR-mimetic drugs with mADF, to assess amplification of beneficial effects. 5) Determine whether biomarker-based outcome measures predict effects on hard clinical outcomes, through longevity studies and studies in disease models (ApoE k.o. and LDL-receptor k.o., dietary-induced obese/insulin resistance, and genetic cancer models in mice). In all studies, different dietary interventions involving intermittent food intake will be compared to classic CR. The central hypothesis is that cyclic changes in intake and metabolism provide signals that turn on a conservation program, without requiring changes in body weight. The availability of biomarkers allows identification of dietary interventions and of metabolic and molecular correlates in an efficient, iterative manner. In summary, our goal is to better understand the metabolic and molecular signals underlying the effects of IF and CR, and to correlate effects on biomarkers with hard outcomes. PUBLIC HEALTH RELEVANCE: The findings that caloric restriction extends lifespan and delays diseases in animal models are remarkable, but have been difficult to translate into human public health benefits for two main reasons: first, because a lifetime of food deprivation and reduced body weight is not practical for most people, and second, because it has been easy to identify the underlying signals responsible for these benefits, when each experiment takes 3 or more years to show that animals live longer. We have identified and will explore here in detail dietary regimens (intermittent fasting alternated with ad-libitum intake) which may not result in weight loss and are likely to be much more feasible for long-term compliance in humans. Our discovery that sensitive, rapid turn-around tests developed in my laboratory may be used as markers of benefit opens the possibility of teasing out the biochemical and molecular signals that underlie the effects of diet, so that drugs which mimic these effects might be developed.